Transgenic non-human animals with expanded mature b cell and plasma cell populations

ABSTRACT

Transgenic non-human animals with expanded mature B cell and plasma cell populations are described, as well as methods of using the transgenic non-human animals to screen for pharmaceutically active agents and to produce targeted antibodies.

TECHNICAL FIELD

The invention relates to transgenic non-human animals that exhibitexpanded mature B cell and plasma cell populations. More particularly,the invention relates to transgenic non-human animals that express oneor more anti-apoptotic polypeptides in the Bcl-2 family, underregulation by an immunoglobulin kappa light chain 3′ enhancer.

BACKGROUND

Multiple myeloma is characterized by the clonal expansion of plasmacells in the bone marrow, and although there are effective therapeuticapproaches, it remains an incurable disease. Difficulties in treatingthis disease include: 1) the resistance of the malignant plasma cells toinduced cell death; 2) significant genetic heterogeneity resulting in afailure to define common genetic events; and 3) lack of good animalmodels. Induction of plasma cell tumors in mice has been achieved byinjection of pristane (mineral oil); however, these tumors are solidplasmacytomas and not bone marrow malignancies. Human fetal boneimplants have been transferred to SCID mice, where the implants supporthuman myeloma cell growth, but the model is highly manipulated in aseverely immunocompromised host.

SUMMARY

The invention is based on the expression of an anti-apoptoticpolypeptide in a transgenic non-human animal. Expression of theanti-apoptotic polypeptide is controlled by a tissue and developmentallyregulated transcriptional enhancer, the immunoglobulin kappa light chain3′ (K3′) enhancer, which restricts expression of the anti-apoptoticpolypeptide to mature B cell/plasma cell populations. As a result, thetransgenic non-human animals exhibit an expansion of mature B cell andplasma cell populations, but no increase in T cell or early B cellpopulations. The transgenic non-human animals also can express aproliferative oncogene such as ras or myc under control of a tissue anddevelopmentally regulated transcriptional enhancer. Transgenic non-humananimals that express both an anti-apoptotic polypeptide and the geneproduct of the proliferative oncogene typically exhibit plasma celltumors.

In one aspect, the invention features a transgenic rodent (e.g., amouse), the nucleated cells of which include a transgene, wherein thetransgene includes an immunoglobulin kappa light chain 3′ enhancersequence operably linked to a nucleic acid sequence that encodes ananti-apoptotic polypeptide in the Bcl-2 family. The transgenic rodentmay exhibit an expanded plasma cell population and an expanded mature Bcell population as compared with a corresponding wild-type rodent. Theanti-apoptotic polypeptide can be selected from the group consisting ofBcl-2, Bcl-xL, Bcl-W, and Mcl-1. A human Bcl-xL polypeptide can beparticularly useful. The transgene further can include a kappa promoteroperably linked to a nucleic acid sequence encoding the anti-apoptoticpolypeptide.

In another aspect, the invention features a transgenic rodent, thenucleated cells of which include (a) a first transgene that includes animmunoglobulin kappa light chain 3′ enhancer sequence operably linked toa nucleic acid sequence encoding an anti-apoptotic polypeptide in theBcl-2 family; and (b) a second transgene that includes a B celldevelopmentally regulated transcriptional enhancer sequence operablylinked to a proliferative oncogene nucleic acid sequence. The transgenicrodent can contain a plasma cell tumor. The proliferative oncogenenucleic acid sequence can be ras or myc. The B cell developmentallyregulated transcriptional enhancer sequence can be an immunoglobulinkappa light chain 3′ enhancer sequence or an immunoglobulin heavy chainenhancer sequence. The anti-apoptotic polypeptide can be selected fromthe group consisting of Bcl-2, Bcl-xL, Bcl-W, and Mcl-1. A human Bcl-xLpolypeptide can be particularly useful.

The invention also features progeny of such transgenic rodents, whereinthe nucleated cells of the progeny include the transgene. In addition,the invention features isolated cells (e.g., plasma cells) of suchtransgenic rodents.

In yet another aspect, the invention features a method for identifyingan agent that inhibits development of a plasma cell tumor. The methodcan include a) administering a test agent to a transgenic rodentdescribed above, wherein the transgenic rodent develops a plasma celltumor in the absence of pharmacological intervention; and b) determiningif the test agent inhibits development of the plasma cell tumor in thetransgenic rodent as compared with a corresponding transgenic rodent towhich the test agent has not been administered.

The invention also features a method for identifying an agent fortreating a plasma cell tumor. The method can include a) administering atest agent to a transgenic rodent described above, wherein thetransgenic rodent exhibits a plasma cell tumor; and b) determining ifthe test agent slows tumor growth, stops tumor growth, reduces tumorsize, or decreases plasma cell number in the transgenic rodent ascompared with a corresponding transgenic rodent to which the test agenthas not been administered.

In another aspect, the invention features a method for producingpolyclonal antibodies. The method can include immunizing a transgenicrodent, the nucleated cells of which comprise a first transgene, thefirst transgene including an immunoglobulin kappa light chain 3′enhancer sequence operably linked to a nucleic acid sequence encoding ananti-apoptotic polypeptide in the Bcl-2 family, wherein the transgenicrodent exhibits an expanded plasma cell population and mature B cellpopulation as compared with a corresponding wild-type rodent; andharvesting the polyclonal antibodies. The transgenic rodent further caninclude a second transgene, the second transgene including a B celldevelopmentally regulated transcriptional enhancer sequence operablylinked to a proliferative oncogene nucleic acid sequence, wherein thetransgenic rodent exhibits a plasma cell tumor.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic depiction of the bcl-xL transgene. “3′KE” refersto 3′ kappa enhancer and “KP” refers to kappa promoter.

FIG. 2A is a graph showing isotype specific Ig levels in the serum ofBcl-XL mice and littermate controls (LMC). *, p<0.05; #, ng/ml. FIG. 2Bis a pair of graphs showing TNP-Ficoll and TNP-CGG specificimmunoglobulin levels in Bcl-XL and LMC mice. *, p<0.05.

FIG. 3A is a line graph showing survival curves of double transgenic,single transgenic, and LMC mice. FIG. 3B is a column graph showingsurvival of cultured splenocytes from double transgenic, singletransgenic, and LMC mice.

DETAILED DESCRIPTION

In general, the invention provides transgenic non-human animals thatcontain an anti-apoptotic transgene in their nucleated cells. Expressionof the anti-apoptotic transgene can be regulated by, for example, theκ3′ enhancer, which is active late in B cell development. The transgenicnon-human animals provided herein typically exhibit properties useful inthe study of multiple myeloma, including an expansion of mature B celland plasma cell populations, and an increase in immunoglobulin levels.Transgenic non-human animals with such properties can be used todetermine whether or not a particular agent is useful for treating orpreventing multiple myeloma. In addition, transgenic non-human animalswith such properties can be used for producing targeted polyclonalantibodies.

Transgenic Non-Human Animals

As used herein, “transgenic non-human animal” includes foundertransgenic non-human animals as well as progeny of the founders, progenyof the progeny, and so forth, provided that the progeny retain thetransgene. Transgenic non-human animals can be, for example, farmanimals such as pigs, goats, sheep, cows, horses, and rabbits, rodentssuch as rats, guinea pigs, and mice, and non-human primates such asbaboons, monkeys, and chimpanzees. Transgenic rodents (e.g., transgenicmice) are particularly useful.

Tissues and cells (e.g., B cells or plasma cells) obtained from thetransgenic non-human animals also are provided herein. The nucleatedcells of the transgenic non-human animals provided herein contain atransgene that includes a nucleic acid sequence encoding ananti-apoptotic polypeptide in the Bcl-2 family. The nucleic acidsequence encoding the anti-apoptotic polypeptide can be a cDNA or caninclude introns or adjacent 5′- or 3′-untranslated regions (e.g., agenomic nucleic acid). As used herein, the term “anti-apoptoticpolypeptide in the Bcl-2 family” refers to any chain of amino acids,regardless of post-translational modification, that has the ability topromote cell survival and that contains at least one of the four (e.g.,one, two, three, or four) Bcl-2 homology. (BH) domains (BH1, BH2, BH3,and/or BH4). BH domains represent consensus sequences of anti-apoptoticpolypeptides. See, PROSITE PDOC00829 from the Pfam Protein FamiliesDatabase (Bateman et al. (2002) Nucleic Acids Res. 30:276-280). Aminoacid substitutions, deletions, and insertions can be introduced into aknown BH domain and the resulting polypeptide is an “anti-apoptoticpolypeptide in the Bcl-2 family” provided that the polypeptide retainsthe ability to promote cell survival.

Suitable mammalian Bcl-2 family members include the Bcl-2, Bcl-xL,Bcl-W, Mcl-1, A1/BFL-1, BOO/DIVA, and NR13 polypeptides. See, e.g.,Gross et al. (1999) Genes Dev. 13:1899-1911; and Cory and Adams (2002)Nature Reviews 2:647-656. Nucleic acid sequences encoding Bcl-2, Bcl-W,Mcl-1, or Bcl-xL polypeptides are particularly useful. Bcl-xL refers tothe long isoform of Bcl-x, which typically is found in high levels inpre-B cells and in lower levels as the B cells mature. GenBank AccessionNos. L35049 and Z23115 L20121 provide the sequences of the mouse andhuman bcl-xL cDNAs, respectively.

The nucleic acid sequence encoding the anti-apoptotic polypeptide in theBcl-2 family can be operably linked to a κ3′ enhancer sequence. See,Fulton and Van Ness (1993) Nucleic Acids Res. 21:4941-4947 for adescription of κ3′ enhancer elements. The nucleic acid sequence of theκ3′ enhancer sequence can be found, for example, in GenBank underAccession No. X15878 (Meyer and Neuberger (1989) EMBO J. 8:1959-1964).As used herein, “operably linked” refers to positioning of a regulatoryelement relative to a nucleic acid sequence encoding a polypeptide insuch a way as to permit or facilitate expression of the encodedpolypeptide. In the transgenes disclosed herein, for example, the κ3′enhancer can be positioned 3′ or 5′ relative to the nucleic acidencoding the anti-apoptotic polypeptide, and can be positioned withinthe transgene in either the 5′ to 3′ or the 3′ to 5′ orientation.Typically, the 5′ end of the κ 3′ enhancer is positioned less than 5 kb(e.g., 0 to 1 kb, 1 to 2 kb, 2 to 3 kb, 3 to 4 kb, or 4 to 5 kb) fromthe nucleic acid sequence encoding the anti-apoptotic polypeptide.

Transgenes of the invention can include additional regulatory elements,including, without limitation, promoters, inducible elements, or otherupstream promoter elements, operably linked to the nucleic acid sequenceencoding the polypeptide. In some embodiments, a tissue specificpromoter is operably linked to the nucleic acid sequence encoding theanti-apoptotic polypeptide. Suitable tissue specific promoters canresult in preferential expression of a nucleic acid transcript in cellsof the B cell lineage and include, for example, the kappa V-regionpromoter and the kappa germline promoter. See, Fulton and Van Ness(supra) for a description of the kappa promoters. In other embodiments,a promoter that facilitates the expression of a nucleic acid moleculewithout significant tissue- or temporal-specificity can be used. Forexample, the β-globin promoter can be used, as well as viral promoterssuch as the herpes virus thymidine kinase (TK) promoter, the SV40promoter, or a cytomegalovirus (CMV) promoter.

In some embodiments, the transgene can include a tag sequence thatencodes a “tag” designed to facilitate subsequent manipulation of theencoded polypeptide (e.g., to facilitate localization or detection). Tagsequences can be inserted in the nucleic acid sequence encoding theanti-apoptotic polypeptide such that the encoded tag is located ateither the carboxyl or amino terminus of the anti-apoptotic polypeptide.Non-limiting examples of encoded tags include green fluorescent protein(GFP), glutathione S-transferase (GST), and Flag tag (Kodak, New Haven,Conn.)

Various techniques known in the art can be used to introduce transgenesinto non-human animals to produce founder lines, in which the transgeneis integrated into the genome. Such techniques include, withoutlimitation, pronuclear microinjection (U.S. Pat. No. 4,873,191),retrovirus mediated gene transfer into germ lines (Van der Putten et al.(1985) Proc. Natl. Acad. Sci. USA 82:6148-1652), gene targeting intoembryonic stem cells (Thompson et al. (1989) Cell 56:313-321),electroporation of embryos (Lo (1983) Mol. Cell. Biol. 3:1803-1814), andin vitro transformation of somatic cells, such as cumulus or mammarycells, followed by nuclear transplantation (Wilmut et al. (1997) Nature385:810-813; and Wakayama et al. (1998) Nature 394:369-374). Forexample, fetal fibroblasts can be genetically modified to express ananti-apoptotic polypeptide, and then fused with enucleated oocytes.After activation of the oocytes, the eggs are cultured to the blastocyststage. See, for example, Cibelli et al. (1998) Science 280:1256-1258.Standard breeding techniques can be used to create animals that arehomozygous for the anti-apoptotic transgene from the initialheterozygous founder animals. Homozygosity is not required, however, asthe phenotype can be observed in hemizygotic animals (see the Examplesherein).

Once transgenic non-human animals have been generated, expression of ananti-apoptotic polypeptide can be assessed using standard techniques.Initial screening can be accomplished by Southern blot analysis todetermine whether or not integration of the transgene has taken place.For a description of Southern analysis, see sections 9.37-9.52 ofSambrook et al., 1989, Molecular Cloning, A Laboratory Manual, secondedition, Cold Spring Harbor Press, Plainview; N.Y. Polymerase chainreaction (PCR) techniques also can be used in the initial screening. PCRrefers to a procedure or technique in which target nucleic acids areamplified. Generally, sequence information from the ends of the regionof interest or beyond is employed to design oligonucleotide primers thatare identical or similar in sequence to opposite strands of the templateto be amplified. PCR can be used to amplify specific sequences from DNAas well as RNA, including sequences from total genomic DNA or totalcellular RNA. Primers typically are 14 to 40 nucleotides in length, butcan range from 10 nucleotides to hundreds of nucleotides in length. PCRis described in, for example PCR Primer: A Laboratory Manual, ed.Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995.Nucleic acids also can be amplified by ligase chain reaction, stranddisplacement amplification, self-sustained sequence replication, ornucleic acid sequence-based amplified. See, for example, Lewis (1992)Genetic Engineering News 12:1; Guatelli et al. (1990) Proc. Natl. Acad.Sci. USA 87:1874-1878; and Weiss (1991) Science 254:1292-1293.

Expression of a nucleic acid sequence encoding an anti-apoptoticpolypeptide (e.g., a Bcl-xL polypeptide) in the tissues of transgenicnon-human animals can be assessed using techniques that include, withoutlimitation, Northern blot analysis of tissue samples obtained from theanimal (e.g., bone marrow or spleen tissue), in situ hybridizationanalysis, Western analysis, immunoassays such as enzyme-linkedimmunosorbent assays, and reverse-transcriptase PCR (RT-PCR). Asdescribed herein, expression of Bcl-xL can result in tissue restrictedexpression of the transgene in mature B cell/plasma cell populations,and one or more of the following characteristics: an expansion of Bcells in the marrow, an increase in post switched B cells (multipleisotypes, including IgG, IgA, and IgE), an increase in the plasma cellcontent in the spleen, an increase in IgG⁺ cells in the marrow, bloodlymphocytosis, increased IgH and IgL serum content, and proteinuria. Thekidney may have hyaline tubular casts, foci of abnormal plasma cells,and glomerular amyloid deposition secondary to Ig accumulation. In someembodiments, the transgenic animals can exhibit bone fractures, an eventthat is secondary in plasma cell neoplasia It is understood that aparticular phenotype in a transgenic animal typically is assessed bycomparing the phenotype in the transgenic animal to the correspondingphenotype exhibited by a control non-human animal that lacks thetransgene.

In one embodiment, the transgenic non-human animals further includes asecond transgene that contains a nucleic acid sequence of aproliferative oncogene such as myc or ras. The nucleic acid sequence ofhuman myc, for example, has GenBank Accession No. X00364. The nucleicacid sequence of human Nras has GenBank Accession No. NM_(—)002524. Thenucleic acid sequence of the proliferative oncogene also can be operablylinked to a tissue and/or developmentally regulated transcriptionalenhancer. For example, the nucleic acid sequence of human myc or ras canbe operably linked to the κ 3′ enhancer or to the μ heavy chain intronenhancer. The μ heavy chain enhancer is active early in B celldevelopment and throughout all remaining developmental stages of the Bcell (Gillies et al. (1983) Cell 33:717-728; Banerji et al. (1983) Cell33:729-740; and Grosschedl and Baltimore (1985) Cell 41:885-897). Thesecond transgene also can include other regulatory elements as discussedabove (e.g., a tissue-specific promoter).

Expression of a proliferative oncogene gene product and ananti-apoptotic polypeptide can enhance tumor pathology in a transgenicnon-human animal as compared with a control transgenic non-human animalexpressing only the anti-apoptotic polypeptide. “Control” typicallyrefers to the same background strain and same species of transgenicnon-human animal, e.g., both transgenic non-human animals are rats orboth are mice. As described herein, transgenic mice expressing a myc orras gene product and an anti-apoptotic polypeptide (e.g., a Bcl-xLpolypeptide) can exhibit clonal expansion and tumor formation (e.g.,plasma cell tumors such as bone marrow malignancies or solidplasmacytomas). Plasma cells or mature B cells removed from suchtransgenic mice can have an increased longevity in culture (e.g., thecells may be immortalized). Such cells can be used to, for example,screen for pharmaceutically active agents.

A transgenic non-human animal expressing both an anti-apoptoticpolypeptide in the Bcl-2 family (e.g., a Bcl-xL polypeptide) and aproliferative oncogene can be produced by, for example, crossing (a) atransgenic non-human animal overexpressing an anti-apoptotic polypeptidewith (b) a transgenic non-human animal overexpressing the product of theproliferative oncogene. Transgenic mice overexpressing myc are describedin, for example, Schmidt et al. (1988) Proc. Natl. Acad. Sci. USA85:6047-6051, and Adams et al. (1985) Nature 318:533-538, whiletransgenic mice overexpressing ras are described in, for example,Cardiff et al. (1993) Am. J. Path. 142:1199-1207. Alternatively, asingle line of transgenic non-human animals can be produced by initiallypreparing the non-human animals using the appropriate transgenes.

Antibody Production

Transgenic animals described herein can be used to produce polyclonalantibodies against a protein of interest. In general, a polypeptide ofinterest can be produced recombinantly, by chemical synthesis, or bypurification of the native protein, and then used to immunize atransgenic non-human animal of the invention. Adjuvants can be used toincrease the immunological response, depending on the species oftransgenic non-human animal. Suitable adjuvants can include, forexample, Freund's adjuvant (complete and incomplete), mineral gels suchas aluminum hydroxide, surface-active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin (KLH), and dinitrophenol. Polyclonal antibodies areheterogeneous populations of antibody molecules that are specific for aparticular antigen (e.g., a polypeptide of interest), and can beharvested from the sera of immunized animals (e.g., immunized transgenicanimals containing an anti-apoptotic transgene in their nucleatedcells).

Once produced, antibodies or fragments thereof can be tested for bindingaffinity to the protein of interest by standard immunoassay methodsincluding, for example, enzyme linked immunosorbent assay (ELISA)techniques or radioimmunoassays. See, e.g., Short Protocols in MolecularBiology, Chapter 11, Green Publishing Associates and John Wiley & Sons,ed. Ausubel et al., 1992.

Screening for Pharmaceutically Active Agents

The transgenic non-human animals described herein can be used to screenfor (a) pharmaceutically active agents that inhibit the development ofplasma cell tumors, or (b) agents that can be used for treating plasmacell tumors (e.g., reducing plasma cell number, slowing or stoppingtumor growth, or reducing tumor size). For example, in embodiments inwhich agents are screened for treatment of plasma cell tumors, acandidate agent can be administered to a transgenic non-human animalthat has developed the tumors, and plasma cell number, tumor size, orother characteristics can be monitored in the transgenic non-humananimal and compared with the same characteristics of a correspondingtransgenic non-human animal to which the test agent has not beenadministered.

Suitable candidate agents can include, for example, chemical compounds,mixtures of chemical compounds, biological macromolecules (e.g.,polypeptides), or biological materials such as extracts of bacteria,plants, fungi, and animals. A variety of techniques can be used forrandom and directed synthesis of a wide variety of organic compounds andbiomolecules, including synthesis of randomized oligonucleotides andoligopeptides. Natural or synthetically produced libraries and compoundscan be modified using chemical or physical techniques known in the art,and such techniques can be used to produce combinatorial libraries.Known pharmacological agents may be subjected to directed or randomchemical modifications, (e.g., acylation, alkylation, esterification, oramidification) to produce structural analogs.

Agents can be formulated into pharmaceutical compositions by admixturewith pharmaceutically acceptable non-toxic excipients or carriers andadministered to the transgenic non-human animals by any route ofadministration. For example, parenteral routes such as subcutaneous,intramuscular, intravascular, intradermal, intranasal, inhalation,intrathecal, or intraperitoneal administration, and enteral routes suchas sublingual, oral, or rectal administration can be used;

A number of methods can be used to determine whether or not an agentalters a particular phenotype exhibited by a transgenic non-humananimal, including, without limitation, biochemical, histological, orbehavioral assays. Suitable biochemical assays (e.g., fluorescenceactivated cell sorting (FACS) analysis, ELISA assays) are known in theart. Histological assays also can be used to determine whether on not anagent affects a particular phenotype exhibited by a transgenic non-humananimal (e.g., hyaline tubular casts or foci of abnormal plasma cells inthe kidney).

It is understood that when comparing phenotypes to assess the effects ofa test agent, a statistically significant difference indicates that thatparticular test agent, test dosage, or test duration warrants furtherstudy. Typically, a difference in phenotypes is considered statisticallysignificant at p<0.05 with an appropriate parametric or non-parametricstatistic, e.g., Chi-square test, Student's t-test, Mann-Whitney test,or F-test.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Production and Characterization of Bcl-XL TransgenicMice

Transgenic mice were generated to overexpress BC-X_(L) in theirB-lymphoid cells. To prepare the transgene vector, the 3′ kappa enhancerand V_(κ)21E promoter (3′KE/KP) were isolated from K3′E.KP.LUC (Fultonand Van Ness (1994) Nucl. Acids Res. 22:4216-4123). The Eμ and TKpromoter were excised from a Bcl-XL construct containing murine Bcl-XLcDNA linked to sequences encoding the FLAGS epitope (Fang et al. (1996)Immunity 4:291-299) and were replaced with the 3′KE/KP sequence. Inaddition, a human growth hormone (hGH) minigene sequence (Anderson etal. (1993) EMBO J. 12:1671-1680) was placed at the 3′ end of theconstruct to provide RNA splicing and processing. The resultingp3′KE/13cl-XL transgene construct (FIG. 1) was purified by CsClcentrifugation, linearized with Not I and Ase I, and injected into malepronuclei of FVB/N mouse embryos.

Mice were maintained in standard pathogen free facilities and analyzedbetween the ages of 4-10 weeks. Southern blotting was used to identifyfounders and estimate transgene copy number. Genomic DNA was isolatedfrom tailsnips was obtained as previously described (O'Brien et al.(1997) Mol. Cell. Biol. 17:3477-3487). Proteinase K (20 mg/mL) was addedto the lysis solution, and the samples were incubated overnight at 55°C. to ensure complete sample digestion. Ten jig of genomic DNA wasdigested with Bgl II (Invitrogen Life Technologies, Carlsbad, Calif.),and a Bcl-XL cDNA was used as a probe for Southern blotting. Todetermine clonality of isolated samples, 10 μg of genomic DNA wasdigested with Bam HI (Invitrogen Life Technologies) and probed with DNAcorresponding to the kappa locus.

For genotyping, 100 ng of genomic DNA was amplified by PCR using thefollowing primer sets: Bcl-XL—FLAG, 5′-GACTACAAGGACGACGATGACAAG-3′ (SEQID NO: 1), and RBCLDOWN, 5′-AGTGGATGGTCAGTGTCTGGTCAC-3′ (SEQ ID NO:2);c-Myc—1MYC, 5′-CAGCTGGCGTAATAGCGAAGAG-3′ (SEQ ID NO:3), and 2MYC5′-CTGTGACTGGTGAGTACTCAACC-3′ (SEQ ID NO:4); IL-2—1IL-2,5′-CTAGGCCACAGAATTGAAAGATCT-3′ (SEQ ID NO:5), and 2IL-25′-GTAGGTGGAAATTCTAGCATCATCC-3′ (SEQ ID NO:6).

These analyses revealed that five independent transgenic founders wereproduced. Of the five founders, four demonstrated germline transmissionof the transgene, and three founder lines were maintained andcharacterized. All three of the characterized founder lines demonstratedsimilar phenotypes. All experiments were performed with heterozygousanimals.

To characterize transgene expression, RNA and protein were isolated fromthe marrow, spleen, liver, kidney, lung, heart, lymph, and thyroid of asix-week-old BCl-XL mouse. RT-PCR was used to detect expression of thetransgene in the various tissues. RNA samples were isolated usingTRIzol® and were treated with DNase I (Invitrogen Life Technologies).cDNA was synthesized using Superscript™ RT (Invitrogen, Rockville, Md.),and template cDNA was amplified using the BC1-XL primers used forgenotyping. Actin primers were used as a control (ACTINUP,5′-CCTAAGGCCAACCGTGAAAAG-3′, SEQ ID NO:7; and ACTINDOWN5′-TCTTCATGGTGCTAGGAGCCA-3′, SEQ ID NO:8). In addition, western blottingwas performed as previously described (Liu et al. J. Immunol.165:7058-7063 (2000)) using the following antibodies: anti-Bcl-XL (SantaCruz Biotech, Santa Cruz, Calif.), anti-FLAG (Sigma, St. Louis, Mo.),anti-actin (Santa Cruz Biotech), and anti-mouse Ig (Amersham, Cleveland,Ohio).

Since the transgenic Bcl-XL was tagged with the FLAG® epitope, bothendogenous and transgenic Bcl-XL levels were determined. TransgenicBcl-XL levels were highest in the marrow, spleen, and lymph nodes. Lowlevels of transcripts also were detected in other tissues, likely due totransgene-expressing lymphocytes in peripheral circulation that migratedto those locations, including microscopic foci of lymphocytes that weredetected upon histopathological examination. Western blotting revealedthat transgenic Bcl-XL expression at the protein level was confined tothe marrow, spleen, and lymph. Two isoforms of Bcl-XL wereobserved—Bcl-XL, and the alternatively spliced Bcl-XΔTM isoform (loss oftransmembrane domain; Fang et al. (1994) J. Immunol. 153:4388-4398).

FACS was used in conjunction with RT-PCR to determine which populationsof lymphocytes exhibited 3′KE activity. The following monoclonalantibodies were used for cell staining: anti-CD-3, anti-B220,anti-CD138, anti-IgM, anti-IgD, and anti-HSA (all antibodies from BDPharmingen, San Diego, Calif., except anti-HSA, which was obtained fromT. Waldschmidt, University of Iowa). Standard flow cytometric analysiswas carried out on a FACSCalibur machine (BD Pharmingen), and data werecollected and analyzed using Cellquest Pro (BD Pharmingen). Stainedcells were sorted on a FACSVantage machine (BD Pharmingen).

Marrow cells from Bcl-XL mice were stained with anti-kappa, anti-B220,anti-IgM-FITC (surface) and/or anti-IgM-PE (cytoplasmic) antibodies.Spleen cells from Bcl-XL mice were stained with anti-B220, anti-CD3,anti-IgM, and/or anti-IgD antibodies. Transgene transcripts weredetected in the B220⁺/kappa⁻, B220⁺/kappa⁺, and cIgM^(hi)/sIgM^(lo)marrow populations. Transgenic transcripts were detected in theB220⁺/CD3⁻, IgM^(hi)IgD^(hi)B220⁺ and IgM^(lo)Ig^(hi)B220⁺ spleenpopulations but not detected in the B220⁻/CD3⁻ or IgM^(hi)IgD^(lo)B₂₂₀ ⁺spleen populations. This analysis demonstrated that 3′KE activity andtransgene expression were detectable in bone marrow plasma cells and inB-cells, including immature/transitional B-cells of the spleen, but thattransgene expression was not detectable in T-cells.

To determine whether the Bcl-XL transgenic mice had altered lymphocytecompartments, samples of peripheral blood from age-matched Bcl-XL mice(n=3) and LMC animals (n=2) were analyzed on a Hemavet® automated cellcounter. All Bcl-XL animals demonstrated lymphocytosis compared to LMC,with an average count of 13.2±2.26 K/μL versus 3.5±0.21 K/μL (P=0.02).

To further characterize the differences in the lymphocyte populations,samples of cells from LMC or Bcl-XL mice were stained withfluorescently-labeled antibodies corresponding to different B- andT-lymphocyte cell surface markers, and analyzed by flow cytometry.Splenocytes from age-matched LMC and Bcl-XL mice were stained withanti-B220 and anti-CD3 antibodies. The percentages of B- andT-lymphocytes were similar in both groups. Since the 3′KE is notstrongly active until the immature and mature stages of B-celldevelopment, altered lymphocyte populations were not expected untilthose stages and beyond. Indeed, no differences in the BP-1⁺ pre-B cellpopulations were observed between Bcl-XL and LMC mice. Marrow cells fromBcl-XL transgenic and LMC mice were stained with anti-B220, anti-IgM,and anti-HSA antibodies, and spleen cells from Bcl-XL transgenic and LMCmice were stained with anti-B220, anti-IgD, and anti-IgM antibodies. TheBcl-XL transgenic animals showed a significant increase in theB220⁺/IgM^(hi)/IgD^(hi) immature/transitional B-cells of the spleen, aswell as the B220⁺/IgM⁺ immature (HSA^(high)) and mature (HSA^(low))B-cells of the marrow. Marrow cells also were stained with anti-B220 andanti-CD138 antibodies to examine the plasma cell (PC) compartments ofLMC and Bcl-XL transgenic mice. No significant differences were observedbetween the CD138^(hi)B220^(lo)PC populations of the two groups.

To evaluate serum immunoglobulin levels, serum samples were collectedfrom Bcl-XL and LMC mice for western blotting with an anti-mouse Igantibody. These studies demonstrated that total amounts of heavy andlight chain Ig proteins were significantly elevated in the Bcl-XL miceas compared to LMC, to the extent that Ig proteins were excreted in theurine of Bcl-XL mice. ELISAs were conducted as previously described(Burns et al. (2003) Bone Marrow Transplant. 32:177-186) to determineisotype-specific serum Ig levels. Bcl-XL animals demonstratedsignificantly (P<0.05) elevated levels of the IgM, IgG1, IgG2b, IgA, andIgE isotypes as compared to LMC (FIG. 2A). Serum samples were analyzedby serum protein electrophoresis and two-dimensional gelelectrophoresis, and although the gamma globulin fractions in the Bcl-XLmice were expanded, no clonal spikes were observed in any of thesamples.

Given the putative apoptosis-resistant B-lymphocytes and elevated serumIg levels in the Bcl-XL animals, Bcl-XL animals were examined toevaluate their response to antigenic challenge and to determine whetherthey make more antigen-specific Ig than LMC. BCl-XL and LMC animals wereimmunized with either TNP-Ficoll, a T-cell independent antigen, orTNP-CGG, a T-cell dependent antigen. Primary immunizations were given incomplete Freund's adjuvant (CFA), and secondary immunizations were givenfourteen days later in incomplete Freund's adjuvant (IFA). Serum sampleswere collected fourteen days after the secondary immunization, a timepoint when maximum antibody production was expected. ELISAs wereperformed to estimate antigen-specific Ig levels. These experimentsrevealed that the Bcl-XL mice produced 65% more TNP-Ficoll specific Igthan LMC (P<0.05), although Bcl-XL and LMC animals produced similaramounts of TNP-CGG specific Igs (FIG. 2B). Thus, Bcl-XL mice producedore T-cell independent antigen-specific Ig than LMC.

For histological analyses, mice were euthanized, necropsied, and fixedin 10% formalin (Sigma, St. Louis, Mo.). Selected tissues (kidneys,spleen, lymph nodes, liver, lung, heart, gastrointestinal tissue, andlong and flat bones) were embedded in paraffin and sectioned at 3-5 μm.Tissue sections were either stained with hematoxylin and eosin orprepared for immunohistochemical analysis (IHC). For IHC, tissuesections were steamed for 30 minutes in 1 mM EDTA, pH 8.0, and blockedwith 3% hydrogen peroxide, Avidin/Biotin Block (Vector, Burlingame,Calif.), and Dako Protein Block (Dako, Carpenteria, Calif.). Thesections were incubated overnight with anti-B220 or anti-CD138antibodies (BD Pharmingen) at 4° C., and then incubated with StrepavidinHRP enzyme conjugate (Dako, Carpenteria, Calif.) for 15 minutes. VectorNova Red Substrate (Vector, Burlingame, Calif.) was used for colordetermination. Sections were counterstained with Mayer's hematoxylin.

Hematoxylin and eosin staining revealed no abnormalities in the LMC micestudied. In contrast, histologic analysis of tissue sections from Bcl-XLmice revealed profound lymphocytic pathology in the three founder linesstudied, and the incidence of microscopic lesions correlated directlywith transgene copy number and age of the animal. Thesehistopathological changes were detected in all Bcl-XL mice studied,ranging between 3 and 24 months of age.

The Bcl-XL mice demonstrated pathological changes in the kidney, andmice that had urine Ig levels detectable by Western blot analysisdemonstrated the most pronounced pathology. Furthermore, a number ofanimals developed perivascular foci of lymphocytes in the kidney.Immunostaining of kidney sections with anti-CD138 or anti-B220antibodies showed that the foci stained intensely for surface CD138.butdid not stain for surface B220, consistent with the cell surfacephenotype of PCs. The presence of PC foci in the kidney and in othertissues indicated that the total amount of Ig protein filtered by thekidney was increased in the transgenic animals. As a result, hyalinerenal tubular casts (Ig protein deposits in renal tubules) were detectedin a number of animals. In addition, foci of abnormal plasma cells(including Mott cells) with atypical nuclei were observed in renaltubules of transgenic animals; the cells found in these foci wereconsistent with PC neoplasia. Two of the transgenic animals withextensive renal pathology also exhibited glomerular amyloid deposition,as determined by Congo red stain.

Histopathological changes also were noted in the marrow and otherlymphoid organs of the Bcl-XL mice. Evaluation of bone marrow sectionsrevealed that while marrow architecture was intact, the percentage oflymphocytes comprising the marrow cavity was significantly increased ascompared to LMC. Additionally, nests of PCs were found in the marrow ofmice greater than one year of age.

Radiographs of multiple mice showed a lack of diffuse demineralizationor focal lesions in the Bcl-XL or LMC mice. Some of the micedemonstrated sheets of PCs in their lymph nodes. Foci of CD 138⁺ PCs andB220⁺ lymphocytes also were detected in other non-lymphoid soft tissues,including liver and lung in BCl-XL animals. Radiographs of mice withconfirmed PC pathology also revealed distinct fractures in the femora(proximal to the femoral head) of two mice and the scapula of one mouse.

Lymph node involvement was unappreciable, except for a single mouse withpopliteal lymph node enlargement (the node contained sheets of plasmacells). In addition, three transgenic mice from two separate founderlines exhibited significant gall bladder distention upon necropsy. Thefluid contained within the gall bladders of these animals containedsignificant amounts of heavy and light chain Ig proteins. Multiple otheranimals demonstrated foci of PCs in multiple soft tissues, includinglung and liver.

Example 2 Production and Characterization of Bcl-XL/c-Myc DoubleTransgenic Mice

The 3′KE/Bcl-XL mouse was crossed with the Eμ/c-Myc mouse to evaluatethe cooperativity of Bcl-XL and c-Myc under control of the 3′KE and Eμpromoters, respectively. Coexpression of anti-apoptotic Bcl-XL andoncogenic c-Myc in the double transgenic animals was highly fatal, and50 percent of the double transgenic mice died of a lymphoproliferativedisorder at 6.7 weeks (FIG. 3A). Mononuclear spleen cells from all fourgenotypes comprising the F1 progeny were cultured in RPMI 1640 mediumsupplemented with 10% FCS, 2 mM L-glutamine, 100 U/mL penicillin, and100 μg/mL streptomycin (all from Invitrogen Life Technologies). Viablecells were counted at days 3 and 7. The majority of cells from singletransgenic mice and LMC were dead at day 7, while more than 60% of thedouble transgenic cells were viable (FIG. 3B). Furthermore, more than60% of splenocytes from the double transgenic mice were still viableafter four weeks in culture.

Samples of peripheral blood from age-matched double transgenic mice(n=2) and LMC mice (n=2) were analyzed on a Hemavet® automated cellcounter. The transgenic animals demonstrated lymphocytosis as comparedto the LMC animals, with an average count of 79.16±3.08 K/μL versus6.32±2.74 K/μL (P=0.02). Staining with anti-kappa and anti-B220antibodies showed that 52.78±6.23 % of the peripheral blood mononuclearcells from the double transgenic mice were B220⁺/kappa⁺, versus5.38±0.24 % of the peripheral blood mononuclear cells from LMC. Inaddition, serum Ig levels were detected by Western blotting. While theserum of double transgenic mice contained more heavy and light chain Igproteins than the serum of LMC or c-Myc mice, the BCl-XL mice had themost elevated levels of serum Ig proteins.

Necropsies revealed that all double transgenic animals demonstrated avery pronounced splenomegaly. In addition, multiple light-colorednodules up to 2 mm in diameter were observed across the surface of thelivers of the double transgenic animals. Histological sections ofkidneys, spleen, lymph nodes, liver, lung, heart, gastrointestinaltissue, thymus, pancreas, and long and flat bones were stained withhematoxylin and eosin. These studies revealed an infiltration ofmononuclear cells with large, round to polygonal euchromatic nuclei anda low mitotic rate in the kidneys, spleen, lymph node, liver, lung,heart, thymus, and pancreas. Similar mononuclear cells were seen in thesternum. Radiographs of the mice showed osteolytic lesions in the longbones of the double transgenic animals. When these areas of bone weresectioned and stained, they were found to contain solid PC tumors. Thetrabecules within the bones were lysed, revealing that the marrowcavities were filled with solid high-grade tumors. In some of the bones,the tumor cells penetrated the corticalis and entered the surroundingsoft tissue. Southern blot analysis was used to examine the clonality ofsamples isolated from two double transgenic mice. Clonally-relatedpopulations were detected in samples of blood, marrow, spleen, and livernodule.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A transgenic rodent, the nucleated cells of which comprise atransgene, said transgene comprising an immunoglobulin kappa light chain3′ enhancer sequence operably linked to a nucleic acid sequence encodingan anti-apoptotic polypeptide in the Bcl-2 family, wherein saidtransgenic rodent exhibits expanded plasma cell and mature B cellpopulations as compared with a corresponding wild-type rodent.
 2. Thetransgenic rodent of claim 1, wherein said transgenic rodent is a mouse.3. The transgenic rodent of claim 1, wherein said anti-apoptoticpolypeptide is selected from the group consisting of Bcl-2, Bcl-xL,Bcl-W, and Mcl-1.
 4. The transgenic rodent of claim 1, wherein saidanti-apoptotic polypeptide is a human Bcl-xL polypeptide.
 5. Progeny ofthe transgenic rodent of claim 1, wherein the nucleated cells of saidprogeny comprise said transgene.
 6. An isolated cell of the transgenicrodent of claim
 1. 7. The cell of claim 6, wherein said cell is a plasmacell.
 8. The transgenic rodent of claim 1, wherein said transgenefurther comprises a kappa promoter operably linked to a nucleic acidsequence encoding said anti-apoptotic polypeptide.
 9. A transgenicrodent, the nucleated cells of which comprise: (a) a first transgenecomprising an immunoglobulin kappa light chain 3′ enhancer sequenceoperably linked to a nucleic acid sequence encoding an anti-apoptoticpolypeptide in the Bcl-2 family; and (b) a second transgene comprising aB cell developmentally regulated transcriptional enhancer sequenceoperably linked to a proliferative oncogene nucleic acid sequence,wherein said transgenic rodent contains a plasma cell tumor.
 10. Thetransgenic rodent of claim 9, wherein said proliferative oncogenenucleic acid sequence is ras.
 11. The transgenic rodent of claim 9,wherein said proliferative oncogene nucleic acid sequence is myc. 12.The transgenic rodent of claim 9, wherein said B cell developmentallyregulated transcriptional enhancer sequence is an immunoglobulin kappalight chain 3′ enhancer sequence.
 13. The transgenic rodent of claim 9,wherein said B cell developmentally regulated transcriptional enhancersequence is an immunoglobulin heavy chain enhancer sequence.
 14. Thetransgenic rodent of claim 9, wherein said anti-apoptotic polypeptide isselected from the group consisting of Bcl-2, Bcl-xL, Bcl-W, and Mcl-1.15. The transgenic rodent of claim 9, wherein said anti-apoptoticpolypeptide is a human Bcl-xL polypeptide.
 16. Progeny of the transgenicrodent of claim 9, wherein said progeny comprise said first transgeneand said second transgene.
 17. An isolated cell of the transgenic rodentof claim
 9. 18. The cell of claim 17, wherein said cell is a plasmacell.
 19. A method for identifying an agent that inhibits development ofa plasma cell tumor, said method comprising: a) administering a testagent to a transgenic rodent, the nucleated cells of which comprise (i)a first transgene comprising an immunoglobulin kappa light chain 3′enhancer sequence operably linked to a nucleic acid sequence encoding ananti-apoptotic polypeptide in the Bcl-2 family; and (ii) a secondtransgene comprising a B cell developmentally regulated transcriptionalenhancer sequence operably linked to a proliferative oncogene nucleicacid sequence, wherein said transgenic rodent develops a plasma celltumor in the absence of pharmacological intervention; and b) determiningif said test agent inhibits development of said plasma cell tumor insaid transgenic rodent as compared with a corresponding transgenicrodent to which said test agent has not been administered.
 20. A methodfor identifying an agent for treating a plasma cell tumor, said methodcomprising: a) administering a test agent to a transgenic rodent, thenucleated cells of which comprise (i) a first transgene comprising animmunoglobulin kappa light chain 3′ enhancer sequence operably linked toa nucleic acid sequence encoding an anti-apoptotic polypeptide in theBcl-2 family; and (ii) a second transgene comprising a B celldevelopmentally regulated transcriptional enhancer sequence operablylinked to a proliferative oncogene nucleic acid sequence, wherein saidtransgenic rodent exhibits a plasma cell tumor; and b) determining ifsaid test agent slows tumor growth, stops tumor growth, reduces tumorsize, or decreases plasma cell number in said transgenic rodent ascompared with a corresponding transgenic rodent to which said test agenthas not been administered.
 21. A method for producing polyclonalantibodies, said method comprising immunizing a transgenic rodent, thenucleated cells of which comprise a first transgene, said firsttransgene comprising an immunoglobulin kappa light chain 3′ enhancersequence operably linked to a nucleic acid sequence encoding ananti-apoptotic polypeptide in the Bcl-2 family, wherein said transgenicrodent exhibits an expanded plasma cell and mature B cell population ascompared with a corresponding wild-type rodent; and harvesting saidpolyclonal antibodies.
 22. The method of claim 21, wherein saidtransgenic rodent further comprises a second transgene, said secondtransgene comprising a B cell developmentally regulated transcriptionalenhancer sequence operably linked to a proliferative oncogene nucleicacid sequence, wherein said transgenic rodent exhibits a plasma celltumor.